Engineering of a mammalian VMAT2 for cryo-EM analysis results in non-canonical protein folding

Vesicular monoamine transporter 2 (VMAT2) belongs to the major facilitator superfamily (MFS), and mediates cytoplasmic monoamine packaging into presynaptic vesicles. Here, we present two cryo-EM structures of VMAT2, with a frog VMAT2 adopting a canonical MFS fold and an engineered sheep VMAT2 adopting a non-canonical fold. Both VMAT2 proteins mediate uptake of a selective fluorescent VMAT2 substrate into cells. Molecular docking, substrate binding and transport analysis reveal potential substrate binding mechanism in VMAT2. Meanwhile, caution is advised when interpreting engineered membrane protein structures.


Significance
It is a good point that protein engineering for structure determination should be used with caution, and the successful generation of the ~4-Angstrom map of wild-type frog VMAT2 by single particle cryo-EM is technically impressive.However, the publications describing experimentally-determined apo, substrate-, and inhibitor-bound human VMAT2 structures that were noted and analyzed by the authors (Refs 15 -18) greatly diminished the significance of the structural work presented here.

Data and methodology
The authors clearly did a great deal of work to identify VMAT2 proteins amenable to cryo-EM structure determination including screening expression and stability of the protein from twelve species.They attempted to solve the cryo-EM structure of the wild-type protein, but initially failed due to the small size and featureless structure of the detergent solubilized protein.The authors then generated VMAT2 constructs with BRIL or AmpC inserted into various loops to increase the size and enhance the shape of the molecule to aid in particle alignment.One of the sheep VMAT2-BRIL constructs, OaVMAT2TM8/9-BRIL, was used for single particle cryo-EM data collection and structure determination that resulted in a 3.2-Angstrom map.However, the map revealed a dimeric structure with an unexpected monomer fold and an unexpected dimer interface based on comparison with a previous structure of a ref 11).The authors went on to carefully analyze the substrate and inhibitor binding properties of OaVMAT2TM8/9-BRIL and performed computational docking to determine whether the unexpected fold of the cryo-EM structure was consistent with the properties of the WT protein or an artifact of the BRIL insertion.The authors concluded that the engineered protein was misfolded.The authors then shifted their focus back to cryo-EM analysis of WT type VMAT2 proteins (human, sheep, and frog) and, remarkably, obtained a 4-Angstrom reconstruction of frog VMAT2, XIVMAT2, that revealed the structure of the TM domain and some loops.This apo structure was found to be in the lumen-facing conformation and the authors describe a cytosolic gate comprised of three methionine and one tyrosine residue.The authors computationally docked substrate (serotonin) and inhibitor (reserpine) to the cryo-EM structure and describe the details of the binding sites.They use the substrate and inhibitor docking results to mutate and biochemically validate the binding sites in human VMAT2, and their mutagenesis and binding studies supported the identities of the binding site residues.The authors compared their apo XIVMAT2 structure with the recently solved substrate-and inhibitorbound structures of human VMAT2 and found that their structure was similar to serotonin-bound VMAT2 (Refs 17 and 18).They also found that their computational docking of serotonin was supported by the serotonin-bound cryo-EM structures.However, they found that the docking results with the inhibitor reserpine were significantly different from a cryo-EM structure of reserpine-bound human VMAT2 (with MBP and MBP-specific DARPin fused to the N-and C-termini respectively) (Pidathala et al. Ref 16).The quality and thoroughness of the work, analysis, and presentation are excellent and may be of general interest to technical structural biologists.However, a substantial portion of the manuscript describes analysis of the mis-folded OaVMAT2TM8/9-BRIL which is only valuable as a cautionary tale (as intimated by the manuscript title) and the remainder focuses on the apo XIVMAT2 structure that has been superseded by higher-resolution structures of human VMAT2 in a variety of mechanistically revealing structural states.

Analytical approach
The analyses were thorough and considered available relevant data.

Suggested improvements
The authors could try to solve the cryo-EM structures of wild-type serotonin-and reserpine-bound XIVMAT2 (or preferably human VMAT2) to determine whether the protein engineering used in Refs 16 and 17 altered the structures.

Clarity and context
The manuscript was well written and presented and includes analysis of work published during manuscript preparation.References Appropriate references were included and discussed.
Reviewer #2 (Remarks to the Author): The work by Lyu et al. presents two cryo-EM structures of VMAT2, featuring BRIL-incorporated sheep VMAT2 in an atypical dimer form, and frog VMAT2 in a lumen-facing conformation adopting a canonical MFS fold.The authors also use molecular docking and MST binding experiments to provide some insights into the mechanisms of serotonin and reserpine, although these insights do not significantly extend beyond what has been previously shown in published structural/functional works on human VMAT2.This work may still be useful to the field of VMAT2.However, some more experiments are needed to strengthen the paper.I have the following suggestions: 1.It is important to demonstrate the transport activity of the constructs compared to the wild type, particularly the OaVMAT2TM8/9-BRIL variant, as substrate binding does not always correlate with transport activity.If the construct is not functional, it is hard to see value in analyzing the atypical MFS structure.It is well known that construct modification can lead to adverse effects, including changes in protein fold.For example, a single amino acid mutation in TRPV5 (PMID: 28878326) can change its fold from swapping to non-swapping.For VMAT2, it is true that other groups do use engineered constructs, but they all demonstrated that the constructs used have transport activity.
2. The authors stated that they also studied wild-type OaVMAT2 (line 112) but did not disclose its oligomeric state.It is important for the authors to clarify whether the wild-type is a monomer or a dimer.If the wild-type is a monomer, it should be clearly stated that the dimerization is a pure artifact, instead of using vague language like "hypothesize" or "may" in the sentence, "We hypothesize that replacement of the TM8/9 loop with BRIL may have altered OaVMAT2 folding." 3. The fused BRIL looked far away from the dimerization interface.How does it contribute to the dimer formation?Or does the author know if it is the fused BRIL or the deletion of the linker that induces dimerization?4. In line 58, the authors mentioned that the AmpC-incorporated construct (OaVMAT2TM8/9-AmpC) is also stable for cryo-EM analysis.Does this construct also form a dimer? 5. What is the sequence conservativity of the dimerization interface?If the residues mediating dimerization are conserved, why was the dimerized form not observed in frog VMAT2 or in reported cases (human VMAT2)?This should be mentioned and discussed.
6. What is the rationale for using different SEC buffers for sheep (Tris pH 7.5) and frog (MES pH 6.0) VMAT2?The buffer pH change may induce different conformations for proton-driven transporters.Are the different conformations observed in sheep and frog VMAT2 caused by the pH?This should be addressed.
7. Why was the MES pH 6.0 buffer used for the MST measurement?Is the binding consistent between MES 6.0 and Tris 7.5 buffers? 8. Reserpine inhibits VMAT2 in its cytosol-facing conformation.Could the authors elaborate on the reason why they used a lumen-facing model (frog VMAT2) to do docking analysis for reserpine?experimentally determined structures to gain insight into substrate and inhibitor binding.They described similarities and differences between their docking results and related experimentally determined co-structures.

Response:
Thanks for your comment.

Significance
It is a good point that protein engineering for structure determination should be used with caution, and the successful generation of the ~4-Angstrom map of wild-type frog VMAT2 by single particle cryo-EM is technically impressive.However, the publications describing experimentally-determined apo, substrate-, and inhibitor-bound human VMAT2 structures that were noted and analyzed by the authors (Refs 15 -18) greatly diminished the significance of the structural work presented here.

Response:
Thanks for your comment.Indeed, it's unfortunate that our first VMAT2 structure (OaVMAT2TM8/9-BRIL) was in an atypical fold, which took us more time to address related issues.
Certainly, it's a bit disappointing to lose a race.But we understand that's how science advances, and we are making our best effort to contribute to this process.

Reviewer #2 (Remarks to the Author):
The 1.It is important to demonstrate the transport activity of the constructs compared to the wild type, particularly the OaVMAT2TM8/9-BRIL variant, as substrate binding does not always correlate with transport activity.If the construct is not functional, it is hard to see value in analyzing the atypical MFS structure.It is well known that construct modification can lead to adverse effects, including changes in protein fold.For example, a single amino acid mutation in TRPV5 (PMID: 28878326) can change its fold from swapping to non-swapping.For VMAT2, it is true that other groups do use engineered constructs, but they all demonstrated that the constructs used have transport activity.

Response:
Thanks for your insightful comment and suggestion.In the revised manuscript, we have employed a cell-based uptake assay to measure transport activities of various VMAT2 constructs (Fig. 1g and Table S3).Interestingly, OaVMAT2TM8/9-BRIL showed ~62% of transport activity compared to wild-type OaVMAT2 (Fig. 1g and Table S3), suggesting that OaVMAT2TM8/9-BRIL is a (partially) functional MFS transporter with an atypical fold.Certainly, evaluation of the biological relevance of this atypical fold of OaVMAT2TM8/9-BRIL requires further investigation.Meanwhile, this data further suggests that extra caution may be necessary when interpreting structures of engineered proteins.This description has been added in the Results and Discussion sections.
2. The authors stated that they also studied wild-type OaVMAT2 (line 112) but did not disclose its oligomeric state.It is important for the authors to clarify whether the wild-type is a monomer or a dimer.If the wild-type is a monomer, it should be clearly stated that the dimerization is a pure artifact, instead of using vague language like "hypothesize" or "may" in the sentence, "We hypothesize that replacement of the TM8/9 loop with BRIL may have altered OaVMAT2 folding."

Response:
Thanks for your comment and question.The wild-type OaVMAT2 appears a monomer in detergent solutions as revealed by a chemical crosslinking experiment (no dimer band) (Fig. S1e).In the original manuscript, the sentence "We hypothesize that replacement of the TM8/9 loop with BRIL may have altered OaVMAT2 folding" was meant to describe different positions of TM11 and TM12 (compared to a canonical MFS fold, Fig. S4d) that may be caused by the insertion of BRIL in the TM8/9 loop.And as a result, TM11 and TM12 (together with TM5 and TM8) participated directly in formation of the dimerization interface between two OaVMAT2TM8/9-BRIL monomers (Fig. 1a).As suggested, we have added description about the oligomeric state of wild-type OaVMAT2 in the revised manuscript (Discussion section) and edited the text to clearly state that the oligomeric state of wild-type OaVMAT2 is different from that of OaVMAT2TM8/9-BRIL, which is likely an artifact induced by its atypical fold and engineering (Discussion section).
3. The fused BRIL looked far away from the dimerization interface.How does it contribute to the dimer formation?Or does the author know if it is the fused BRIL or the deletion of the linker that induces dimerization?

Response:
Thanks for your comment and question.Indeed, the fused BRIL is located quite far away from the dimer interface.Therefore, it looks less likely that the fused BRIL (or the deletion of the TM8/9 loop) caused dimerization directly.Rather, in our opinion, insertion of BRIL in the TM8/9 loop may have caused different placement of TM11 and TM12 in OaVMAT2TM8/9-BRIL compared to a canonical MFS fold (Fig. S4d).As a result, TM11 and TM12 (together with TM5 and TM8) participated directly in formation of the dimerization interface between two OaVMAT2TM8/9-BRIL monomers (Fig. 1a).Therefore, the fused BRIL may have contributed indirectly to the dimer formation through TM11 and TM12.4. In line 58, the authors mentioned that the AmpC-incorporated construct (OaVMAT2TM8/9-AmpC) is also stable for cryo-EM analysis.Does this construct also form a dimer?

Response:
Thanks for your question.Based on chemical crosslinking experiments, a minor dimer band of OaVMAT2TM8/9-AmpC in detergent solutions was observed in SDS-PAGE analysis, whereas the majority of OaVMAT2TM8/9-AmpC shows in the monomer band (or in the high-molecular-weight aggregate band) (Fig. S1e).The chemical crosslinking data has been added in the Discussion section.
5. What is the sequence conservativity of the dimerization interface?If the residues mediating dimerization are conserved, why was the dimerized form not observed in frog VMAT2 or in reported cases (human VMAT2)?This should be mentioned and discussed.

Response:
Thanks for your question and suggestion.The residues that form the dimer interface of OaVMAT2TM8/9-BRIL are highly conserved among different species (residues highlighted in yellow in Fig. S8).However, in a canonical MFS fold, many of these residues would not be exposed on the protein surface due to different positions of TM11 and TM12.For example, in XlVMAT2WT, residues Y417, Y421, A424 and F428 on TM11 (equivalent to Y421, Y425, A428 and F432 in OaVMAT2) are facing the interior of VMAT2 and are not available for dimerization (Fig. S9b).This result may explain why OaVMAT2TM8/9-BRIL forms a dimer but XlVMAT2WT does not.This description has been added in the Discussion section.
6. What is the rationale for using different SEC buffers for sheep (Tris pH 7.5) and frog (MES pH 6.0) VMAT2?The buffer pH change may induce different conformations for proton-driven transporters.Are the different conformations observed in sheep and frog VMAT2 caused by the pH?This should be addressed.

Response:
Thanks for your question and suggestion.The VMAT2 samples were prepared in different pH buffers intentionally, trying to capture different conformations of VMAT2.For example, frog VMAT2 (XlVMAT2) was actually prepared in both pH 6.0 (mimicking the vesicular lumen pH) and pH 7.5 (mimicking the cytosol pH) buffers for cryo-EM data collection, and we were hoping to solve two different conformations with these two pH conditions.But unfortunately, we were only able to solve the XlVMAT2 structure at ~4 Å with the pH 6.0 data set.
work by Lyu et al. presents two cryo-EM structures of VMAT2, featuring BRIL-incorporated sheep VMAT2 in an atypical dimer form, and frog VMAT2 in a lumen-facing conformation adopting a canonical MFS fold.The authors also use molecular docking and MST binding experiments to provide some insights into the mechanisms of serotonin and reserpine, although these insights do not significantly extend beyond what has been previously shown in published structural/functional works on human VMAT2.This work may still be useful to the field of VMAT2.However, some more experiments are needed to strengthen the paper.I have the following suggestions: